Impaired antigen receptor induced calcium mobilization in a phospholipase C-gamma1 deficient B cell line

B lymphocytes express phospholipase C-γ₁ (PLC-γ₁) and phospholipase C-γ₂ (PLC-γ₂) isozymes. However, the relative importance of these two isozymes in B cell signaling is not known. We report here the identification and analysis of a B cell line deficient in PLC-γ₁. Mature splenic B lymphocytes and a panel of cell lines representing pre-B, immature and mature B cell stages expressed phospholipase C-γ (PLC-γ), but not the β or δ isoforms of phospholipase C (PLC). While all the tested B cell lines and primary splenic B cells expressed PLC-γ₁ and PLC-γ₂ isozymes, the L1.2 B cell line exclusively expressed PLC-γ₂, but not PLC-γ₁ isozyme. The PLC-γ₁ deficient L1.2 B cells expressed levels of surface IgM comparable to that of PLC-γ₁ positive 70Z/3 B cell line. However, stimulation through the antigen receptor induced Ca⁺⁺ response in 70Z/3, but not L1.2 B cells, suggesting a requirement for PLC-γ₁ in antigen receptor induced Ca⁺⁺ signaling in these cells. The possible use for L1.2 cells in testing the regulation of PLC-γ₁ and PLC-γ₂ dependent calcium mobilization in B lymphocytes is discussed.     

Inhibition of proliferation of a murine myeloma cell line and mitogen-stimulated B lymphocytes by the antibiotic amphotericin B (Fungizone).

Amphotericin B at the concentration normally used for routine suppression of fungal infection in tissue culture strongly inhibits the proliferation of NS1/1 myeloma cells and the LPS-induced activation of B lymphocytes from mouse spleen. The proliferation of T lymphocytes induced by concanavalin A (Con A) was less affected by the antibiotic, indicating that B-lymphocyte proliferation was preferentially inhibited. The unexpected sensitivity of B-lymphoid cells to amphotericin B precludes its use as an anti-fungal agent in the production of hybridomas from fusions between these cells.     

Heat-regulated expression of the hepatitis B virus surface antigen in the human Wish cell line

The DNA fragment coding for the hepatitis B virus surface antigen (HBsAg) was placed under the control of a human 70 kDa heat-shock protein (hsp70) promotor sequence. This plasmid construct has been used in transfection experiments to establish a stable amnion cell line of human origin (Wish), expressing an HBsAg in a heat-regulated fashion. Post-translational modifications, such as assembly, glycosylation, secretion and production of both major and middle S proteins appear to function normally. In addition, production of HBsAg under various protocols of heat induction is described. After inoculation into nude mice, development of tumours has been observed at the site of injection. Tumour cells, dispersed by means of collagenase or trypsin treatment from excised tumours, and subsequently seeded into Petri dishes, were able to secrete the same quantities of HBsAg after heat induction as were cells of the original cell line.     

Joining-chain (J-chain) negative mice are B cell memory deficient

The systemic immune response of joining-chain (J-chain)-deficient mice (J(-/-) mice) on a C57BL/6 background against the hapten 4-hydroxy-3-nitrophenyl (NP) was analysed. While primary IgG responses to the hapten were similar to those observed in WT control animals, secondary immune responses were compromised both at the level of serum IgG and the number of responding B cells. The repertoire switch from lambda to kappa in secondary immune responses was diminished in J(-/-) mice. The number of somatic mutations introduced in the V(H) 186.2 gene during the primary immune response was reduced, while the frequency of affinity-increasing mutations in position 33 was similar. By adoptive transfer experiments it could be shown that the compromised secondary immune response was transferred with T cells from J(-/-) mice. Thus, J-chain-deficient mice have a selective defect in T helper cell function during B cell immune responses, resulting in a deficiency in the formation of B cell memory.     

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.     

Human helper T cell lines established by coculture of normal human cord leukocytes with an HTLV-II-infected rabbit leukocyte cell line (Ra-IIA).

Three helper T cell lines, designated CR-IIA (CR-IIA-1, CR-IIA-2, and CR-IIA-3), were established by coculturing normal human cord leukocytes with a lethally irradiated HTLV-II (human T-lymphotropic virus type II)-infected rabbit leukocyte cell line (Ra-IIA). CR-IIA had a normal human karyotype and expressed the surface markers CD3(+), CD4(+), CD8(-), CD19(-), CD25(+) and HLA-DR(+), confirming their helper T cell nature. CR-IIA cells were all free of Epstein-Barr virus nuclear antigen and were immunoreactive with serum samples from HTLV-I- or HTLV-II-infected patients and with anti-HTLV-I, p19 or p24 antibody. The provirus genome of HTLV-II was detected in these cell lines by the polymerase chain reaction combined with a digoxigenin-enzyme-linked immunosorbent assay. Electron microscopy of CR-IIA-1 cells revealed a few immature type C virus particles. These results suggest that HTLV-II was transmitted from the infected rabbit leukocyte cell line to human cord helper T lymphocytes with the development of immortalized HTLV-II-producing T cell lines.     

High level expression of apoptosis inhibitor in hepatoma cell line expressing Hepatitis B virus

The serious result of hepatitis B (HBV) virus infection is development of hepatocellular carcinoma (HCC). However, the reason of development of HCC in HBV infected patients is still unclear. Recently, the suppression of cell apoptosis is found to relate with the development of cell carcinogenesis, therefore, the expression of apoptosis inhibitor in the virus related cancer line such as hepatoma cell line HepG2.215 was investigated. There are at least six Human apoptosis inhibitors (IAP) have been identified now. They are cIAP1, cIAP2, XIAP, NAPI, survivin and pIAP. Using gene-assay technology, we have recently compared the expression of IAPs in the HepG2.215 cells that persistently expresses Hepatitis B virus by integrated HBV genome with its parent cell line HepG2. The results suggest that there was obviously increase of cIAP2 and cIAP1 in the HepG2.215 cells versus HepG2 cells. Those observations imply a possibility of long time HBV infection could induce the over-expressing apoptosis inhibitors, furthermore, causing the liver cancer. The high expression of cIAP1 and cIAP2 in HBV expressing cells was confirmed by RT-PCR and Northern blot analysis. However, we did not find the change of NIAP and suvivin in HepG2.215 cells. In contrast, the expression of XIAP was down in the HepG2.215 cells comparing with HepG2 cells. How HBV triggers the over-expression of apoptosis inhibitor is unclear. Transient transfection of HepG2 cells with the plasmids expressing different HBV proteins such as S, M, L, X and core proteins did not give a decisive conclusion. Further study is going on now.     

Ontogeny of the antibody-forming cell line in mice. III. The generation of mature anti-sheep red blood cell-specific B cells is antigen-dependent.

A role for antigen in the generation of fully mature splenic type B cells has been shown. In adoptive transfer experiments, cells from bone marrow or fetal liver required a longer period to give an anti-sheep red blood cell plaque-forming cell (PFC) response than those from spleen. This delay was not overcome by allowing the cells a 7-day sojourn in the irradiated host before antigen challenge. A two-stage protocol was designed in which the in vivo generation of fully mature cells could be measured by their ability to give PFC in lipopolysaccharide-stimulated cultures in vitro. These experiments showed that a critical factor which influences the final differentiation of bone marrow or fetal liver cells into mature, splenic type B cells is exposure to antigen.     

Differentiation of Clara cell (distal type) antigen in human fetal bronchial epithelial cell line (HFBE).

A human fetal bronchial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibroblastic shape, however, they still maintained an immuno-reactivity to cytokeratin, carcinoembryonic antigen (CEA) and epithelial membrane antigen (EMA). When grown on a collagen gel in a growth hormone-supplemented medium, their spindle shape became more conspicuous. With the additional supplement of 6 micrograms/ml vitamin A, most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive to periodic acid Schiff (PAS) and barely positive to alcian-blue (AB) staining. Electron microscopy revealed well-developed rough endoplasmic reticulum (rER), enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted products containing glycoconjugates of two different molecular weights. The higher molecular weight-product was identified as hyaluronic acid and the lower molecular weight-product as a mixture of neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. While the secretion of hyaluronic acid was inhibited by vitamin A in a dose-dependent manner, that of the neutral glycoproteins was most enhanced by vitamin A in the range from the physiological concentration of 600 ng/ml to 6 micrograms/ml. A monoclonal antibody (SEC-41) generated against the secretory products with the lower molecular weight detected a glycoprotein of approximately 52 kd in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intra-cytoplasmic secretory granules in these cells. When tested on freeze-sectioned lung tissue, immunohistochemical reactivity of SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung and respiratory epithelial cells of the fetal lung tissue. Moreover, this antibody could detect the secretory glycoproteins in the broncho-alveolar lavages (BAL) of two human cases. This paper has clearly demonstrated that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and under appropriate culture conditions they can be stimulated to undergo differentiation into a Clara cell type.     

Cytoplasmic bridges and gap junctions in an insect cell line (Aedes albopictus).

Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used study simultaneously the diffusional and electrical properties of intercellular junctions. Diffusion studies involved injection of fluorescent molecules into one cell of a cell pair and visual inspection of their intercellular redistribution. Electrical measurements involved a dual voltage clamp method and whole-cell recording with patch pipette. The voltage clamp protocol was aimed at examining the dependency of the junctional conductance, gj, on membrane potential, Vm. Cell pairs exhibiting a voltage-dependent gj were found to allow intercellular diffusion of Lucifer Yellow CH (molecular mass, 443 Da), but not of FITC-dextran (molecular mass, 4,400 Da). This response pattern is consistent with the presence of gap junctions in the intercellular junctions. Cell pairs showing no voltage dependence of gj were found to permit intercellular diffusion of both Lucifer Yellow CH and FITC-dextran (dextran labelled with fluorescein isothiocyanate). This behaviour is compatible with the presence of cytoplasmic bridges connecting the two adjacent cells. Hence, in culture the cells investigated express two kinds of intercellular structures, gap junctions and cytoplasmic bridges.     

Variants of the mucosal mast cell line (RBL-2H3) deficient in a functional membrane glycoprotein.

We have isolated and characterized subpopulations of the rat mucosal mast cell line, RBL-2H3, carrying either high or low density of a glycoprotein, recently established as mast cell function-associated antigen (MAFA, Ortega et al., 1991), on their surface. These populations were investigated in order to better define the involvement of the MAFA in coupling the immunological stimulation of mast cells to mediator release. The MAFA density on the cell surface of the deficient subpopulation was less than or equal to 10-20% that of the parental population and this phenotype was found to be stably maintained for several months. In contrast, the MAFA-enriched cells had maximally twice the number of copies per cell surface than that of the parental population and this phenotype was less stable. Significantly, low copy number of MAFA on the cell's surface was accompanied by a markedly different secretory response, i.e. (i) a considerable decrease in the secretory response to the Fc epsilon RI-mediated stimulus (ii) a marked enhancement of the ionomycin induced secretion. In order to gain insight into the causes for this decrease in cellular response to the Fc epsilon RI-mediated stimulus, we measured the amplitudes of several biochemical processes which are assigned to the stimulus-secretion coupling cascade. The Fc epsilon RI-mediated uptake of 45Ca2+ by the MAFA-deficient cells was considerably lower than that of the parental and MAFA-enriched cells. Similarly, these cell's Fc epsilon RI-induced rise in [Ca2+]i (both the initial transient as well as the sustained elevation), was markedly lower than that of the parental line and the MAFA-enriched cells. Moreover, the low initial transient rise in [Ca2+]i was found to be correlated with the decrease in Fc epsilon RI-mediated IP3 levels. We therefore examined the cell's content of the phosphatidyl-inositides hydrolyzing enzyme, phospholipase C gamma 1. This was found to be similar in the parental line and in its derived subpopulations. However, PLC gamma 1 activation, as measured by the time course of phosphorylation of its tyrosines, showed a marked difference: while PLC gamma 1 tyrosine phosphorylation, in the parental cells, was only transient (detected already 1 min after antigen addition and declined afterwards to basal levels at ca. 10 min), in the MAFA-deficient cells, tyrosine phosphorylated PLC gamma 1 was also observed 1 min after antigen addition, yet showed no decrease with time in its phosphorylation intensity for up to 30 min.(ABSTRACT TRUNCATED AT 400 WORDS)     

Signal transmission through the B cell-specific MB-1 molecule at the pre-B cell stage.

Specific binding of antigens to the surface immunoglobulin M (sIgM) triggers B cells with several biochemical events involved in receptor-mediated signal transmission for proliferation and differentiation into antibody-producing cells. Recent studies with the Digitonin lysis method identified the sIgM-associated component, IgM-alpha (B34)/Ig-beta, as the possible candidate for the transducer molecule(s) in the immunoglobulin receptor-mediated signal transmission. The 34 kd protein (B34 or IgM-alpha) of this component is suggested to be encoded by the B cell-specific mb-1 gene. We prepared monoclonal antibodies which recognize the mb-1 gene product (MB-1) and studied the functional role of MB-1 in the signal transmission in B lineage cells. Using murine pre-B lymphoma cells (18-81 and 70Z/3), we demonstrated the early phase increase of the intracellular [Ca2+]i concentration and the subsequent inhibition of the proliferation by the monoclonal anti-MB-1 antibody (11-18-5). These results clearly demonstrate signal transmission through the surface MB-1 molecule on B lineage lymphomas. This MB-1-mediated signal transmission in pre-B cell lines would suggest an alternative function of MB-1 acting at the pre-B cell stage.     

Relapsing and remitting experimental autoimmune encephalomyelitis in B cell deficient mice

Experimental autoimmune encephalomyelitis (EAE) is an animal model for the human autoimmune central nervous system (CNS) disease multiple sclerosis (MS). To examine the role of B cells in EAE with a relapsing and remitting disease course (R-EAE) we generated (B10.PL x SJL/J)F1 mice deficient in B cells by disrupting their mu heavy chain transmembrane region (B10.PL x SJL/J)F1 muMT-/-. By immunizing (B10.PL x SJL/J)F1 and (B10.PL x SJL/J)F1 muMT-/- mice with the encephalitogenic N-terminal peptide Acl-11 of myelin basic protein (MBP), we observed that B-cell deficient mice exhibited a relapsing and remitting disease course. Since a similar day of onset and day of first relapse were observed these data suggest that B cells do not play a vital role in the activation of T cells leading to the initiation of EAE, nor in the reactivation of T cells resulting in R-EAE.     

SEA和B7-1基因真核共表达载体的构建及在B16细胞的表达

目的构建葡萄球菌肠毒素A（SEA）和小鼠B7-1基因真核共表达载体。方法采用PCR和RT-PCR方法分别克隆了带B7-1跨膜区的SEA（SEA-B7tm）和小鼠B71基因,中间通过内部核糖体进入位点（Internal ribosome entry site,IRES）序列的连接克隆至真核表达载体pcDNA3.1＋。利用阳离子脂质体将重组质粒转染B16细胞,间接免疫荧光法检测B7-1和SEA分子在B16细胞膜表面的表达情况。结果测序结果与Genebank中公布的SEA、小鼠B7-1 cDNA序列相符,双标记间接免疫荧光检测结果表明B7-1、SEA同时在转染的B16细胞膜上表达。结论成功构建了SEA和小鼠B7-1真核共表达载体,为进一步研究SEA和B7-1联合应用抗肿瘤免疫治疗及其免疫机理奠定了基础。